Structural studies have determined that the predicted Nef protein sequence of HIV-1, HIV-2 and SIV can be dissected into at least three regions: (a) leucine-zipper (dimerization domain); (b) autophosphorylation (kinase domain); and (c) acidic alpha-helical (transcriptional activation domain). Using molecular biological approaches, experiments have been designed aimed at defining functional domains of the Nef proteins of HIV-1 and HIV- 2. Genetic, biochemical and biological studies will be used to analyze the relationship between Nef structure and function. Preliminary results have shown that the HIV-2 (NIH-Z) Nef protein forms stable homo-oligomers in vitro and in vivo, and that phosphorylation appears to favor equilibrium towards the homodimer (50 kD) species. This finding implicates the conserved leucine repeat sequence motif in the oligomerization. In addition, glutathione S-transferase (GST) Nef fusions have been constructed, and have expressed the leucine repeat region of HIV-2 Nef and the acidic transcriptional activation-like region at the carboxy terminus of HIV-1 Nef in E. coli. These GST-Nef fusion proteins are used as Nef-affinity matrices bound to glutathione-Sepharose to identify T cell factors that bind to, and are potential in vivo targets for Nef. Using GAL4-Nef fusions and transient transfection/CAT assays, identification will be made of regions of the HIV-1 Nef protein that have transcriptional activation or repressive activity in COS-7 or HeLa cells. In addition, an endoplasmic reticulum (ER) retention signal sequence (I/VDDL) has been identified at the extreme carboxyl terminus of the HIV-1 Vpu protein. Site-specific mutagenesis was used to remove the terminal dipeptide sequence of the ER signal. Both WT and site-specific nef and vpu mutant genes were introduced by electroporation into H9 cells in the pCDNA-1 Neo vector for subsequent analysis of function and mechanism.